Evolution of DNA replication origin specification and gene silencing mechanisms.

DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

Ultrastructural changes in the lungs of neonatal rats intratracheally inoculated with meconium.

Meconium aspiration syndrome has been for many years an important cause of neonatal respiratory distress in newborn babies and sporadically reported in animals. This investigation was designed to study the ultrastructural and morphometric changes in the lungs of neonatal rats following the intratracheal inoculation of meconium. Seven-day-old Fischer-344 rats (n = 24) were randomly allocated in two groups. One group was intratracheally inoculated with saline solution and the second group received homologous meconium. Neonates were euthanatized at 1, 3 and 7 postinoculation days (PID) and lungs were examined by light and electron microscopy. Saline solution did not induce any ultrastructural changes in the lung. In contrast, meconium induced deciliation, recruitment of neutrophils and pulmonary alveolar macrophages to the bronchoalveolar space, intravascular sequestration of neutrophils and aggregation of platelets at PID 1 and 3. Other ultrastructural changes at PID 1 and 3 included interstitial edema and escape of red cells and fibrin into the alveolar space and interstitium. Interstitial edema and sequestration of neutrophils were responsible for the significant increase in thickness of alveolar septa. At PID 7 there was hyperplasia and enlargement of type II pneumocytes as well as interstitial proliferation of mesenchymal cells with intra-alveolar fibrosis. It was concluded that intratracheal inoculation of meconium in neonatal rats induces acute ultrastructural changes followed by a reparative response.

Microscopic changes induced by the intratracheal inoculation of amniotic fluid and meconium in the lung of neonatal rats.

Meconium aspiration syndrome is a major contributor to neonatal respiratory distress in infants and it has been sporadically recognized in neonatal animals. This investigation was designed to study the short and long term effects of meconium and amniotic fluid in the lungs of neonatal rats. Seven-day-old rats (n = 123) divided in three groups were intratracheally inoculated with saline solution, amniotic fluid or meconium. Rats were euthanatized on 1, 3, 7, 14, 28, 56 and 112 postinoculation days (PID) and the lungs were examined by light microscopy. Saline solution did not induce any change while amniotic fluid elicited only a mild foreign body response which disappeared by PID 14. In contrast, meconium induced an exudative alveolitis characterized by recruitment of neutrophilsn in the bronchoalveolar spaces. Meconium also induced atelectasis, hyperinflation and thickening of alveolar septa all of which had disappeared by PID 14. Starting at PID 7, neutrophils were progressively replaced by macrophages, giant cells, and some fibroblasts. There were sporadic foci of mineralization starting at PID 14 and lasting up to PID 112. Some mineralized foci became lined with cuboidal epithelial cells at PID 28. Meconium was slowly degraded but still evident by PID 112. It was concluded that inoculation of meconium in neonatal rats induces acute microscopic changes typical of meconium aspiration syndrome. The long term lesions induced by meconium consisted of persistent multifocal histiocytic alveolitis and bronchiolitis reaction with occasional foci of calcification.

Measuring sphere-like structures using transrectal ultrasonography.

Sphere-like structures are commonly measured for clinical or research purposes. We measured two diameters, the circumference (perimeter) and cross sectional area of 113 equine ovarian follicles in a water bath then determined the relationships between those measurements and the actual volume of fluid in the follicles. Cross-sectional area, diameter3 and area2/3 were all highly correlated with the volume of the structures and superior (p < 0.05) to diameter or perimeter as predictors of volume. As predictors of volume, there was no significant difference (p > 0.05) between cross-sectional area, diameter3, and area2/3. Because of the ease of measuring diameter, we suggest that diameter3 be used to estimate the actual size of sphere-like structures by ultrasonographic measurement. A table was generated for clinical use to show how much the largest diameter of a fluid-filled structure (follicle, cyst, hematoma, etc.) would have to increase or decrease to indicate a significant change in the volume of the structure.

Ultrastructural and secretory heterogeneity of fa/fa (Zucker) rat islets.

Many previous studies of obese rodents documented biochemical changes in pancreatic islets that contribute to hyperinsulinemia in vivo. Those studies used heterogeneous populations of islets, although the size of islets from obese rats ranges from < 100 to > 500 microm. Here, functional and morphological changes in size-sorted (< 125 and > 250 microm diameter) islets from obese Zucker (fa/fa) rats were correlated. Ultrastructural examination revealed that > 250 microm cultured islets had an increased number of immature secretory granules in the beta cells. The number of degranulated beta cells in > 250 and < 125 microm cultured islets from fa/fa rats was higher than in lean rat islets (33 vs 25%). The glucose EC50 values for cultured islets were 4.64 +/- 0.43, 7.9 +/- 0.70 and 7.29 +/- 1.64 mmol.l(-1) for > 250 microm, < 125 microm, and lean groups, respectively. Inhibition of insulin secretion by 10 mmol.l(-1) mannoheptulose was reduced by 50% in > 250 microm islets compared with small islets. Studies of individual beta cells by reverse hemolytic plaque assay revealed 3-fold more cells from > 250 microm islets were stimulated by 1.4 mmol.l(-1) glucose than cells from < 125 microm islets. We conclude that functional defects in mixed size populations of islets from fa/fa rats are mainly due to alterations in the large islets, whereas smaller islets have relatively normal function. Exposure to high glucose exacerbates morphological and functional differences of large islets, which could have important implications in the transition to noninsulin-dependent diabetes when beta cell insulin production is unable to compensate for hyperglycemia.

Polychlorinated biphenyl congener 153-induced ultrastructural alterations in rat liver: a quantitative study.

Alterations in the liver of male and female Sprague-Dawley rats fed PCB congener 153 (2,2',4,4',5,5'-hexachlorobiphenyl) at 0.5, 5, or 50 ppm concentrations in diets for 13 weeks were determined morphometrically. A dose-dependent increase in hepatocyte volume was detected; the cytoplasmic compartment contributed to the increase in cell volume in an overwhelming fashion. Eighty percent and 250% increase in smooth endoplasmic reticulum volume and its surface area in hepatocytes were estimated in animals of both genders from 5- and 50-ppm groups, respectively; the organelle played the largest part in the increase in cytoplasmic volume. Rough endoplasmic reticulum alteration was shown to depend on gender, where the volume per hepatocyte was augmented by 40% and 45% in females of 5- and 50-ppm groups, respectively, however, 30% and 20% decreases in volume of this organelle were noted in males at those congener concentrations. A decrease of 13% in normal mitochondria volume at 50 ppm concentration was observed, which may have been a consequence of a transformation of these mitochondria to abnormal types. Two types of abnormal mitochondria, named Type I and Type II, were defined: the former comprised mitochondria that had cristae which laying parallel to the long axis of the organelle and the latter showed C- or ring-shaped profiles. Data analysis revealed a trend toward an increase in abnormal mitochondria volume in the cells as the congener concentration elevated. In addition, a threefold increase in the volume of lysosomal elements per hepatocyte was noted in 50 ppm PCB-fed rats of both genders. Also, a significant increase in peroxisome volume per cell in female rats was detected at a lower concentration than it was in the male. This study, which is a first ultrastructural quantitative investigation on the effects of a PCB that included many parameters. The methodology, and the data may prove useful to provide better understanding of pathology in the evaluation and regulation of toxic chemicals.

A small-scale outdoor plant growth chamber with modulated enhancement of solar UV-B radiation.

An outdoor, solar illuminated growth chamber with modulated UV-B enhancement, is described and its performance characterized. The chamber was clad with Teflon film that transmitted both UV-A and UV-B with little absorption and whose transmission did not change significantly over 1 yr. An array of UV-B emitting fluorescent tubes with cellulose diacetate filters provided additional UV-B radiation inside the chamber with little additional UV-A, while a similar chamber with unenergized tubes provided an ambient control. Ambient and enhanced UV-B levels were measured using a pair of integrating broad band radiometers, and it was found that frequency modulation allowed control of lamp intensity over a range in excess of 100:1. Within the UV-B chamber, enhanced levels of integrated UV-B could be maintained at a constant ratio over ambient throughout the day. However, it was not sufficient simply to maintain a constant ratio of radiometer output voltages: the angular responses of the two radiometers had to be well matched. The shape of the UV-B portion of the enhanced spectrum closely followed that expected under ozone depleted skies, while the enhancement of UV-A was insignificant. The spectral profile obtained in the UV-B depended on the thickness of the cellulose diacetate filters used to block short wavelength UV radiation emitted by the fluorescent tubes. The number (thickness) of cellulose diacetate filters required to achieve this match varied with season. Although the filters did degrade with exposure to UV-B it was not necessary to replace them for several months. This was due to: (i) low rate of filter degradation because the lamps were rarely required to be run at full brilliance except at midsummer midday; and (ii) increased tube intensity compensated for reduced filter transmission.

Porcine lingual taste buds: a quantitative study.

BACKGROUND: Taste buds on the pig tongue have been neither studied in detail nor quantified. We have counted the numbers of fungiform and vallate papillae, estimated their taste bud densities, and computed numbers of taste buds per volume of papilla on porcine tongue. Pig was chosen for this work because it is an omnivorous animal, and is one of the better models for study of nutrition-related problems in omnivorous human beings. METHODS: Tongues from three adult pigs were obtained from the local abattoir, fixed in 10% neutral buffered formalin, and mapped for the fungiform and vallate papillae. Serial 15 microns sections were prepared from blocks containing the papillae, and were stained by H & E. Density and volume of the taste buds were estimated using a Bioquant system. RESULTS: Eight hundred fifty and 826 fungiform papillae were observed on tongue I and II, respectively; numerically vallate papillae were 1, 2, and 2 for tongue I, II and III, respectively. Number of taste buds per fungiform papilla was considerably less on the dorsal surface of the tongue (3.88/papilla) than on the lateral side (22.11/papilla). Vallate papillae contained significantly more taste buds, a mean of 732.67 per papilla (t = 14.27; df = 6; P = < 0.05), than in the fungiform papillae. Density of the buds per volume of fungiform papilla was notably less on the dorsum (13.69) than on the lateral border (30.08). The density of vallate papillae taste buds per volume of the papilla was significantly greater than the lateral and dorsal papillae taste bud density (t = 4.28; df = 6; P < or = 0.05). CONCLUSIONS: The present results are relevant to studies on variations of taste sensitivity among regions of the tongue.

Effect of volume variations on osteogenic capabilities of autogenous cancellous bone graft in dogs.

OBJECTIVE: To evaluate the osteogenic effect of differing volumes of autogenous cancellous bone graft (ACBG) placed into partial cortical defects of the ulna. ANIMALS: 15 healthy, mature Beagles. PROCEDURE: Weekly radiographic views of defects were obtained over 8 weeks and were analyzed for bone density by use of radiographic optical densitometry. Histologic sections were obtained 8 weeks after surgery and were planimetrically evaluated for area of total, lamellar, and woven bone. RESULTS: Defects receiving 0.3 and 0.75 g of ACBG had rapid initial bone production, and dogs receiving 0.3 g of ACBG had faster bone ingrowth than did those receiving 0.1 g of ACBG or controls. Defects receiving 0.75 g of ACBG had a rate of bony ingrowth equal to 0.3 g of ACBG. There was no difference in the histomorphometric area fractions of total, lamellar, or woven bone between defects treated with 0.1 or 0.75 g of ACBG, and between grafted or control defects, 8 weeks after surgery. CONCLUSIONS: Overfilling a cortical defect with ACBG does not enhance early osteogenesis within the defect, and underfilling will delay the onset of osteogenesis within the defect. CLINICAL RELEVANCE: A volume of ACBG sufficient only to fill a cortical defect is required to obtain a clinical osteogenic effect from the graft.

Effects of modulatory agents on neurally-mediated responses of trout intestinal smooth musclein vitro.

Mediators and mechanisms responsible for the inhibitory modulation of trout intestinal smooth muscle were examined using a series of putative mediators and substances known to modulate neurotransmission in mammalian systems. Frequency response relationships to transmural stimulation and concentration response relationships to 5-hydroxytryptamine, carbachol, and substance P were established on paired segments of rainbow trout intestinein vitro in the presence and absence of putative modulatory agents. Modulation of neurally-mediated contractions of trout intestine was achieved with dibutyryl cyclic AMP and forskolin, agents that increase intracellular levels of cyclic AMP. The effect appears to be at the level of the smooth muscle, since the adenylate cyclase activator, forskolin, inhibited muscarinic and serotoninergic contractions as well as transmurally stimulated contractions. Substance P-induced contractions were unaffected by forskolin. The endogenous agonists/neurotransmitters which would increase cyclic AMP levels in rainbow trout intestinal smooth muscle are as yet unknown. The effects do not appear to be modulated by vasoactive intestinal peptide (VIP), calcitonin, calcitonin gene-related peptide (CGRP), or agents that activate ß-adrenoceptors. Prostaglandin E2 (PGE2) and α2-adrenergenic agonists are possible agents which will decrease contractility of the smooth muscle. They were only active in the proximal intestine and on transmurally stimulated contractions. The effects of both PGE2 and α2-agonists appear to be prejunctional, decreasing release of contractile neurotransmitters in the enteric nervous system.

Surface coat of sheep pulmonary intravascular macrophages: reconstitution, and implication of a glycosyl-phosphatidylinositol anchor.

BACKGROUND: Pulmonary intravascular macrophages (PIMs) of sheep have a globular surface coat that facilitates endocytosis of tracer particles and Escherichia coli lipopolysaccharide, and is disrupted by the heparin and Brefeldin A treatments. The present study investigated the in vivo dynamics of the coat globules following heparin-mediated removal, and the mechanism of globule organization on the plasma membrane of PIMs in vitro. METHODS: Sheep were administered heparin at a dose of 50 IU/kg body weight IV, and euthanised at 30 min, 3, 6, 12, 48, and 120 hr (n = 2 for each treatment) after the treatment. Control sheep (n = 2) were injected with normal saline solution. The tissues were processed for an ultrastructural examination and acid phosphatase (ACPase) cytochemistry. Heparin-treated lungs were subjected to morphometric analysis of the coat globules. Lung tissues from normal sheep (n = 2) were incubated with phosphatidylinositol-specific-phospholipase C (PIPLC; 2 IU/ml PBS) in vitro for 30 and 75 min. RESULTS: Heparin study: The ultrastructural and morphometric data showed that the coat globules were removed at 30 min and reconstituted within 48 hr of the treatment. The PIMs showed prominent Golgi complexes associated with secretory vesicles, microtubules, and centriole between 3-12 hr of heparin treatment. Acid phosphatase cytochemistry also demonstrated secretory activity in the Golgi complexes of PIMs during the coat reconstitution. PIPLC study: The coat globules of PIMs were removed in a time-dependent mode by the PIPLC treatment without damage to other cell organelles. CONCLUSIONS: This study demonstrates a time-dependent reconstitution of the coat of PIMs in conjunction with secretory activity following heparin-mediated removal, probably through sequestration of the globules from blood. This ability is of functional significance as the coat mediates particle endocytosis by the PIMs. The results also suggest the presence of a glycosyl-phosphatidylinositol (GPI) anchor in tethering of globules on the plasma membrane of PIMs to offer a structural basis for their integrity in pulmonary vascular flow.

Morphological changes in the small intestine of the fetal pig after prenatal stimulation of the sow with ACTH.

Twelve pregnant primiparous sows were catheterized on day 102 of gestation and randomly allocated to receive ACTH (days 112-113 of gestation [2d], days 105-parturition [10d]) or saline. At parturition the 3rd, 4th, 6th and 7th piglet born alive were sacrificed either at birth or at 6 h. The later group was fed bovine colostrum through a stomach tube at 30 min, 2 and 4 h of life. Following sacrifice, the small intestine of each piglet was excised, extended and measured. Piglets from sows in the 2d group tended to be heavier with longer small intestines than either the control or 10d groups. When the small intestine length was expressed as a function of body weight, the 10d group had the greatest ratio, suggesting that the pre-parturition maintenance of elevated cortisol levels either enhanced small intestine growth or attenuated overall weight gain. Two types of enterocyte granules were described at the level of the electron microscope: granular and opaque. Opaque granules from piglets born to sows in the 2d treatment group had both a lower volume fraction and were fewer per unit area when compared to those of either the saline or 10d treatments. The process of macromolecule uptake from the intestinal lumen appeared to have been interfered with as a result of an acute prenatal stimulation with ACTH. The above results suggest that in piglets, the level of circulating cortisol differentially controls the processes of IgG absorption, enterocyte replacement and small intestine growth through separate mechanisms. Further, these processes can be manipulated in the prenatal piglet.

A method for finding stereotaxic coordinates from brain sections.

A method for finding the stereotaxic coordinates of brain areas from actual brain sections is presented. It uses a digitizer connected to a computer to gather coordinates from photographs of brain sections. The coordinates are mathematically translated and rotated to yield stereotaxic atlas coordinates of the areas digitized.

The effects of acute temperature change on smooth muscle contractility of rainbow trout (Oncorhynchus mykiss Walbaum) intestine.

The effects of altered temperature in vivo on in vitro smooth muscle contractility of rainbow trout intestine were investigated. Initial analysis of the data revealed a seasonal variation in the maximal tension of intestinal smooth muscle attainable with 5-hydroxytryptamine (serotonin), carbachol, KCl, and transmural stimulation in vitro. Peaks occurred in spring and troughs in autumn. There was no seasonal cycling of the potency of the stimulants. All data regarding the efficacy of the stimulants were subsequently corrected for seasonal variation. The response of smooth muscle depends on the temperature of the water in which the fish are placed (2°C-20°C). There was a marked linear increase in efficacy and a slight increase in potency of the stimulants with increasing temperature. Changes in responsiveness of the intestinal smooth muscle occurred within 30 min of moving the fish between tanks. Smooth muscle reactivity returned to pretreatment values by 48h. Any changes in responsiveness with regards to time were unlikely to be as a consequence of water temperature, but may have been a result of handling stress.

Changes in smooth muscle contractility of rainbow trout (Oncorhynchus mykiss Walbaum) intestine during acclimation to altered temperature.

The effects of altered water temperature in vivo on in vitro smooth muscle contractility of rainbow trout intestine were investigated. Temperature has a significant effect on receptor-mediated intestinal smooth muscle contractility in the rainbow trout. The efficacy of 5-HT, carbachol, and transmural stimulation increased with temperatures above 10°C, with an optimal increase at 15°C. There was also a modest increase in the potency of 5-HT and carbachol within 2 days of establishing trout at 20°C. By day 8, most of these changes had either stabilized or were returning to control values, suggesting that acclimation changes in membranes and enzyme activities were taking effect. However, the contractile responses to carbachol and transmural stimulation were still increasing at this time. This may imply that the muscarinic receptors are more resistant to membrane acclimation changes and may take longer to adapt. Because these experiments were controlled for handling stress and seasonal changes that affect contractility, we have been able to demonstrate some early changes in smooth muscle contractility that occur during acclimation to altered temperature.

Quantitative regional variation in the expression of major histocompatibility class II antigens in enterocytes of the mouse small intestine.

The qualitative and quantitative expression of major histocompatibility class II antigens was investigated in the absorptive epithelium of the duodenum, jejunum, and ileum from mice of C3H/He (H-2k haplotype) and C57BL/6 (H-2b haplotype) strains by peroxidase-antiperoxidase labelling and image analysis. Immunohistochemical labelling revealed that the expression of class II antigens was greatest in the ileum and decreased proximally towards the duodenum. The villus epithelium of the duodenum showed a granular staining pattern in the apices of some cells. In the jejunum, an increased expression was demonstrated in the apical and basal cytoplasm of all cells covering the villus. Cells at the tip of the villus, in addition, showed staining of the lateral surfaces. Ileal enterocytes demonstrated the most intense immunostaining appearing in the cytoplasm and along baso-lateral surface membranes. Quantitative analyses confirmed that a highly significant (p less than 0.0001) difference in expression of class II antigens occurred in the three regions of the small intestine, which corroborated the qualitative findings. This regional variation of class II molecules by the absorptive epithelium may influence regional differences in antigen presenting functions and immune responsiveness to ingested antigens.

Evaluation of the instantaneous centers of rotation of the stifle before and after repair of torn cruciate ligament by use of the over-the-top technique in dogs.

Seven dogs with torn cruciate ligament were used to evaluate the "over-the-top" repair procedure, using instantaneous center of rotation (ICR). Normal and cruciate-torn stifles of dogs were radiographed before surgery, using an image intensifier videotape system. The injured stifle was reradiographed 6 weeks after surgery. Images of the stifles were photographed from video monitor. Photographs were analyzed by a digitizer hooked to a computer to find the ICR at various angles. Data were analyzed, using multivariate analysis of variance. Relative to those of normal stifles, ICR of the damaged stifles were located significantly (P less than 0.05) proximal and caudal. After surgery, the ICR were still proximal and caudal, but not significantly so. We conclude that the cruciate-torn stifle deviates significantly from normal and that such deviation is restored to normal limits by use of the over-the-top procedure.

Development of the small intestine of piglets in response to prenatal elevation of glucocorticoids.

The effects of prenatal adrenal stimulation and synthetic glucocorticoid supplementation on development of the gastro-intestinal tract of the piglet were investigated. Twelve pregnant sows were treated with either ACTH infusion, Isoflupredone injection or Saline between days 105 and 112 of gestation. Neonatal pigs were weighed, bled and sacrificed at 0 or at 6 h. Piglets sacrificed at 6 h were fed bovine colostrum. Transverse sections were prepared from the duodenum, jejunum and ileum for measurement of the villus amplification factor (VAF) and basal membrane circumference. Sows in the ACTH group showed an elevation in cortisol in response to infusion; this decreased after infusion and then rose again at parturition. Piglets from both the ACTH and Saline groups had more villus surface area per unit of body weight (BW) than those born to Isoflupredone-treated animals. The BW of the ACTH piglets was lower (P less than 0.05) than those of piglets in the other groups. When the weight of the stomach and the Small Intestine (SI) was expressed as a function of the body weight, the stomach and SI:BW ratio was larger (p less than 0.05) in pigs born to ACTH-treated sows. The circumference of the ileum was larger at 6 h than at 0 h. Control pigs had a higher concentration of bovine IgG at 4 and 6 h (P less than 0.05). Observations of the light microscopic preparations indicated a less organized epithelium in both ACTH and isoflupredone pigs sacrificed at 0 h. Light and EM preparations of ileum from ACTH pigs sacrificed at 6 h, showed an abundance of dark-stained vacuoles, characteristic of IgG-containing structures. These became less evident in piglets from the Isoflupredone group and even less so in the control groups. The consequences of these phenomena in terms of absorptive capacity are discussed.

Statistical evaluation of dendritic growth models.

A mathematical model (Kliemann, W. 1987. Bull. math. Biol. 49, 135-152.) that predicts the quantitative branching pattern of dendritic tree was evaluated using the apical and basal dendrites of rat hippocampal neurons. The Wald statistic for chi 2-test was developed for the branching pattern of dendritic trees and for the distribution of the maximal order of the tree. Using this statistic, we obtained a reasonable, but not excellent, fit of the mathematical model for the dendritic data. The model's predictability of branching pattern was greatly enhanced by replacing one of the assumptions used for the original method "splitting of branches for all dendritic orders is stochastically independent", with a new assumption "branches are more likely to split in areas where there is already a high density of branches". The modified model delivered an excellent fit for basal dendrites and for the apical dendrites of hippocampal neurons from young rats (30-34 days postpartum). This indicates that for these cells the development of dendritic patterns is the result of a purely random and a systematic component, where the latter one depends on the density of dendritic branches in the brain area considered. For apical dendrites there is a trend towards decreasing pattern predictability with increasing age. This appears to reflect the late arrival of afferents and subsequent synaptogenesis proximal on the apical dendritic tree of hippocampal neurons.